Characterization of the enzymatic hydrolysis of acetate from alkylacetylglycerols in the de novo pathway of PAF biosynthesis.
This report describes the partial characterization of the enzymatic activity responsible for the hydrolysis of acetate from 1-alkyl-2-acetyl-sn-glycerol, the immediate precursor in the de novo synthesis of PAF (platelet-activating factor or 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) by Ehrlich ascites cells. The highest acetylhydrolase activity for this neutral lipid was associated with the membrane fractions from Ehrlich ascites cells (greater than 90% of total activity); only a minimal level of activity (less than 10%) was observed in the cytosol which contrasts with the cytosolic site of PAF acetylhydrolase in normal cells. Hydrolysis of 1-[3H]hexadecyl-2-acetyl-sn-glycerol by the membrane fraction at pH 7.5 and 37 degrees C gave apparent values for Km and Vmax of 45 microM and 179 nmol/min per mg protein, respectively. Hydrolysis of acetate from 1-[3H]hexadecyl-2-acetyl-sn-glycerol by the membrane fraction was not affected by 5 mM concentrations of Ca+2, Mg+2 or EDTA, but was significantly inhibited (80% reduction) by 10 mM NaF. Based on differences in both the subcellular distribution and response to inhibition by NaF, the neutral lipid acetylhydrolase does not appear to be the same enzyme that hydrolyzes acetate from platelet-activating factor. In contrast to inhibition of diacylglycerol lipase by p-chloromercuribenzoate and N-ethylmaleimide, we found no significant inhibition of acetate hydrolysis from 1-[3H]hexadecyl-2-acetyl-sn-glycerol by either of these compounds. Also, p-nitrophenyl acetate (a nonspecific esterase substrate) failed to inhibit acetate hydrolysis of 1-[3H]hexadecyl-2-acetyl-sn-glycerol. Our studies of this enzyme would indicate that it may play an important role in regulating the levels of platelet-activating factor synthesized by the de novo pathway via hydrolysis of the immediate precursor of PAF.
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Medical Sciences Division, Oak Ridge Associated Universities, TN 37831-0117.
Smith
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Cress
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Snyder
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D000085:Acetates / Q000378:metabolism*; D000818:Animals; D001792:Blood Platelets / Q000378:metabolism; D002286:Carcinoma, Ehrlich Tumor / Q000378:metabolism; D002478:Cells, Cultured; D006868:Hydrolysis; D008049:Lipase / Q000037:antagonists & inhibitors; D010742:Phospholipid Ethers / Q000378:metabolism*; D010972:Platelet Activating Factor / Q000096:biosynthesis*; D011817:Rabbits; D012969:Sodium Fluoride / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D000085:Acetates; D010742:Phospholipid Ethers; D010972:Platelet Activating Factor; C032521:acetyl glyceryl ether phosphorylethanolamine; D012969:Sodium Fluoride; D008049:Lipase